Genetic variability of populations of the white-eared opossum, Didelphis albiventris Lund 1840 (Didelphimorphia; Didelphidae) in Brazil

ISSN 1678-4375 (Online)

Genetic variability of populations of the white-eared opossum,

Didelphis albiventris

Lund 1840 (Didelphimorphia; Didelphidae)

in Brazil

D. C. Nascimento

, B. A. T. P. Campos

_{, E. C. Fraga}

and M. C. Barros

_*

Maranhão – UEMA, Cidade Universitária Paulo VI, CP 9, São Luís, MA, Brasil

b_{Centro de Estudos Superiores de Caxias – CESC, Laboratório de Genética e Biologia Molecular – GENBIMOL,}

Universidade Estadual do Maranhão – UEMA, Praça Duque de Caxias, s/n, Morro do Alecrim, CEP 65604-380, Caxias, MA, Brasil

c_{Programa de Pós-graduação em Biodiversidade, Ambiente e Saúde – PPGBAS, Centro de Estudos Superiores de}

Caxias – CESC, Universidade Estadual do Maranhão – UEMA, Praça Duque de Caxias, s/n, Morro do Alecrim, CEP 65604-380, Caxias, MA, Brasil

*e-mail: mbdene@yahoo.com.br

Received: September 01, 2017 – Accepted: February 10, 2018 – Distributed: November 30, 2019

(With 4 figures)

Abstract

Didelphis albiventris are found throughout Northeast and Central Brazil to central-southern Uruguay and it was subject of few studies in a population level. Given this, the present study investigated the genetic variability of the species using the mitochondrial molecular marker cytochrome oxidase c subunit I. We analyzed samples from the different

biomes within three Brazilian regions: Northeast (Caatinga, Cerrado, and Atlantic Forest), Southeast (Cerrado, Atlantic

Forest, Cerrado/Atlantic Forest, and Cerrado/Caatinga ecotones) and South (Pampa and Atlantic Forest). Software BAPs retrieved five distinct demes: dm 1, dm 2, and dm 5 that occurs in South, Northeast and Southeast regions respectively

and the dm 3 and dm 4 are wide distributed in Northeast and Southeast. Population analysis performed with AMOVA, haplotype network and Mantel test estimated the veracity of the demes. The F_ST shows structuring for the five demes, with dm 1 (South region) isolated from the others, however the other analysis showed the Northeast/Southeast demes (dm 2-5) united, diagnosing gene flow between them, mainly at the transitional zones, in areas as far away as areas with similar latitude interval (Southeast vs South) that was not detected gene flow. In the haplotype network, the mutational

steps was conclusive in split dm1 from dm 2-5 with 15 mutational steps and the Mantel test was moderated, which

is explained by genetic similarity despite the great geographic distances (Northeast/Southeast). Thus, our analysis recognized two different lineages (South and Northeast/Southeast) and indicate that the biomes were not decisive in their isolation. The sharing of demes at the transitional zones and in areas with high latitudinal intervals reflects a recent

ancestral polymorphism for D. albiventris. The plasticity in the occupation of the space by this species contributes in its wide dispersion capability, that is, geographical distribution. Our results revealed important implications for the management of D. albiventris in these transitional zones areas where demes were shared.

Keywords: marsupials, COI, population diversity, geographic lineages.

Variabilidade genética de populações do gambá de orelha branca,

Didelphis albiventris

Lund 1840 (Didelphimorphia; Didelphidae) no Brasil

Resumo

Didelphis albiventris é encontrada em todo o Nordeste e região central do Brasil até o centro-sul do Uruguai e foi alvo de poucos estudos em nível populacional. Dessa forma, o presente estudo, investiga a variabilidade genética da espécie usando o marcador molecular citocromo c oxidase subunidade I. Analisou-se amostras de diferentes biomas de três regiões brasileiras: Nordeste (Caatinga, Cerrado e Floresta Atlântica), Sudeste (Cerrado, Floresta Atlântica,

ecótonos Cerrado/Floresta Atlântica e Cerrado/Caatinga) e Sul (Pampa e Floresta Atlântica). O software BAPs recuperou cinco demes distintos: dm 1, dm 2 e dm 5, que ocorrem nas regiões Sul, Nordeste e Sudeste, respectivamente, e os dm 3 e dm 4, que são amplamente distribuído no Nordeste e Sudeste. Análises populacionais realizadas com AMOVA, rede de haplótipo e teste de Mantel estimaram a veracidade das demes. O F_ST mostrou estruturação para as cinco demes,

1. Introduction

The marsupials of the genus Didelphis Linnaeus, 1758 are a small non-volant mammals that are an essential component of natural communities and good indicators of environmental changes and its degree of conservation (Conde y Vera and Rocha, 2006; Castro, 2012; Quintela et al., 2013). In particular, populations of the white-eared opossum (Didelphis albiventris Lund, 1840) tends to increase in density in response to environmental disturbance (Cáceres et al., 2008), with associated implications for public health given that this species is a natural reservoir for a number of human diseases such as Chagas disease and leishmaniasis (Sherlock et al., 1984; Pinho et al., 2000; De Luca et al., 2003; Bern et al., 2011; Zetun et al., 2014).

Due to its generalist habit, D. albiventris is highly flexible in the use of space, exploiting a variety of vertical strata and occupying large home ranges (Almeida et al., 2008; Sanches et al., 2012). The species is widely distributed in deciduous forests, occurring from northeastern and central Brazil to central and southern Uruguay, as well as in Paraguay, Argentina and Bolivia (Cerqueira and Tribe, 2008). In Brazil there are records in Caatinga, Cerrado, Pantanal, Pampas habitats and transitional zones between these biomes (Cerqueira and Tribe, 2008; Paglia et al., 2012).

Despite the genus Didelphis is common and ecologically successful (Durant, 2001), little is known about the relationships among of D. albiventris populations, with only two studies available: Sousa et al. (2012) and

Rocha et al. (2015). Both works revealed some degree of genetic structuring, the former studied the mitochondrial gene cytochrome c oxidase subunit I (COI) of populations

from southern and southeastern Brazil, and the other the mitochondrial gene cytochrome b from few localities in central Brazil.

Mitochondrial DNA have revealed degree of population structure for many species, being the most used molecular marker for tracing the geographic distribution of genealogical lineages (Avise et al., 1987). Males and females of the D. albiventris present similar patterns of dispersal

(Sanches et al., 2012), and the sex of the specimen should be irrelevant to the selection of the molecular marker, consequently the matrilineal inheritance of the mitochondria should not interfere in the analyses. Notably, mitochondrial genes have been used successfully in population-level and

phylogenetic studies of small mammals (Carvalho et al., 2011; Agrizzi et al., 2012; Sousa et al., 2012; Muller et al., 2013; Rocha et al., 2015). In particular, the COI gene has

been used as a DNA barcode for the identification of a number of different vertebrate groups (Hebert et al., 2003),

and has contributed important findings on D. albiventris

populations (Sousa et al., 2012).

Ecological studies use population and genetic data in attempt to understand the biology of a species. With the scarcity of population genetic information throughout its distribution, especially for the inhabitants of Cerrado and Caatinga, the present work investigated how D. albiventris

populations are distributed in Brazil using the COI as a molecular marker: 1. The species possesses populations structured in the Northeast/South axis? 2. There is more than one population per Biome? 3. If yes, they are shared between Biomes or are exclusive for one Biome? Ecotone

areas has a different role in the population structure of the

species? 4. Is there natural selection or are the populations selectively neutral? 5. In attempt to answer these questions we conducted genetic variability study of populations of the white-eared opossum, D. albiventris.

2. Material and Methods

2.1. Description of the samples

Samples of D. albiventris were obtained from the Northeast, Southeast and South regions of Brazil, between latitudes 04°05’ S and 30º01’ S (Figure 1). A total of 52 samples were obtained in the Northeast region (Paraíba/PB, Sergipe/SE, Pernambuco/PE, Ceará/CE,

Piauí/PI and Maranhão/MA), from the Atlantic Forest,

Caatinga and Cerrado biomes. Eighty four samples from

Southeast (Minas Gerais/MG) from the Atlantic Forest

and Cerrado biomes, and the Cerrado/Atlantic Forest and Cerrado/Caatinga ecotones. Six samples from South

(Rio Grande do Sul/RS) corresponding to the Pampas

and Atlantic Forest (Figure 1; Supplementary Material). The samples from the Northeast were obtained in the Universidade Estadual do Maranhão, for which the collecting data are available in Costa et al. (2012), and

from collaborations with the scientific collections of other

Brazilian research institutions, such as Universidade Federal da Paraíba (UFPB), Universidade Federal de Sergipe (UFS), Universidade Federal do Piauí (UFPI),

and Museu Paraense Emílio Goeldi (MPEG). All samples

áreas com similar intervalo de latitude (Sudeste e Sul), onde não foram detectado fluxo gênico. Na rede de haplótipo,

os passos mutacionais foram conclusivos em separar dm 1 do dm 2-5 com 15 passos mutacionais, e o teste de Mantel

foi moderado, o que é explicado pela similaridade genética apesar da grande distância geográfica (Nordeste/Sudeste). Assim, duas linhagens diferentes (Sul e Sudeste/Nordeste) foram encontradas, indicando que os biomas não foram

decisivos em seus isolamentos. Os compartilhamentos das demes, em zonas de transição e em áreas com elevados

intervalos de latitude, refletem um polimorfismo ancestral recente para D. albiventris. A plasticidade na ocupação do espaço por esta espécie contribui em sua ampla capacidade de dispersão, ou seja, distribuição geográfica. Nossos

resultados revelam importantes implicações para o manejo de D. albiventris nessas áreas de zonas de transição, onde

as demes são compartilhadas.

from Southeast and South were obtained at GenBank from

Sousa et al. (2012).

2.2. DNA extraction, amplification and sequencing DNA was extracted from muscle, liver, and ear tissue using the Phenol-Chloroform protocol of Sambrook and

Russel (2001). We amplified and sequenced a fragment

of the mtDNA COI gene using the primers LCO1490 and HCO2198 (Folmer et al., 1994). The COI gene was

amplified in 25 μl reactions of PCR, consisting of 4.0 μl of dNTP (1.25 nM), 2.5 μl of buffer (10X), 0.5 μl of MgCl2

(25 nM), 0.25 μl of each primer (200 ng/μl), 0.2 μl of Taq DNA Polymerase 10 x 250 μl (GE Healthcare), and 1 μl of template DNA (100 ng/μl), with the final volume being completed by purified water. The samples were amplified in a Veriti 96 well thermal cycler (Applied Biosystems) with an initial denaturation (94ºC for 3 min), 40 cycles of denaturation (94ºC for 45 s), annealing (48ºC for 45 s), and extension (72ºC for 1 min and 30 s), and a final extension

at 72ºC for 3 min. We used the BigDye Terminator v3.1

Cycle Sequencing Kit (Applied Biosystems) for sequencing

reactions. The sequences were obtained using an ABI 3500 automatic DNA sequencer with associated Sequencing

Analysis software (Applied Biosystems).

2.3. Data analysis

The sequences were aligned using Clustal W (Thompson et al., 1994) in BioEdit v7.0.5.2 (Hall, 1999). The analysis of the variable sites was performed in Mega v6.06 (Tamura et al., 2013). Haplotype and nucleotide diversity were estimated in DnaSP v4.9 (Rozas et al., 2003),

and the groups used for the population analyses were defined

using the BAPs software (Corander et al., 2008). In this method, the number of populations is treated as unknown parameter and is directly inferred from the data set without

defining a prior estimate, a dendogram were generated

using Unweighted Pair Group Method using Arithmetic

averages - UPGMA. The populations identified by this

Figure 1. Localities from which the samples were obtained for the present study. The demes were defined according to the

BAPs software and are represented by geometric figures with numbers (designated in Supplementary Material); Biomes are

software were used to structure the subsequent population analyses. The haplotype networks were produced in NetWork v4.6.1.2 (Bandelt et al., 1999). An Analysis of Molecular

Variance (AMOVA) was performed in Arlequin v3.5.2.1

(Excoffier and Lischer, 2010) to obtain the index of allelic

fixation (F_ST). The neutrality tests D (Tajima, 1989) and Fu’s F_S (Fu, 1997) were run in Arlequin v3.5.2.1 (Excoffier and Lischer, 2010). The Mantel test, which relates genetic distance to geographic distance, was run in the Alleles in Space software with 10,000 replications (Miller, 2005).

3. Results

3.1. Molecular characterization and neutrality tests The study included a total of 142 sequences for the mitochondrial COI gene of D. albiventris, made up of a fragment of 655 bps, of which 624 bps were conserved,

31 were variable, and 21 were informative for parsimony analyses, corresponding to 19 haplotypes.

The sequences plotted in the BAPs software (Figure 2)

indicated the existence of five Brazilian demes (dm), as

can be seen in the haplotype network and in the map (Figure 1 e 3). The dm 1 occurs in the Pampas and Atlantic

Forest biomes (South region; H1 and H2), dm 2 in the

Caatinga and Atlantic Forest biomes (Northeast region;

H3-H8) and dm 5 Cerrado, Caatinga, Atlantic Forest

and ecotones (Southeast regions; H16-H19). The dm 3 (H 9-H13) and dm 4 (H14 and H15) are more widely

distributed, being found simultaneously in the Cerrado and Caatinga (Northeast region) and Cerrado, Caatinga,

Atlantic Forest (Southeast region). We can observe that some localities present multiple demes: Xacriabá Amerindian

Reservations/MG that contains dm 3, dm 4 and dm 5; Caxias/MA dm 3 and dm 4, Belo Horizonte/MG and Nova Lima/MG dm 4 and dm 5 (Table 1).

Figure 2. Didelphis albiventris demes defined through the BAPs software for the COI gene. (a) UPGMA tree using the Nei

model. (b) Mixture among the demes obtained, the size of the color bars is proportional to the number of specimens in the

populations.

Figure 3. Haplotype network for the COI gene in the D. albiventris demes. The numbers represents the mutational sites between

The total haplotypic diversity (h) was h = 0.846, and varied from h = 0.387 for dm 5 to h = 0.614 for dm 3,

while total nucleotide diversity (π) was π = 0.00615, and varied from π = 0.00078 for dm 4 to π = 0.00152 for dm

3 (Table 1). No significant p values were found in any deme for the D test (Tajima, 1989), and a significant value for Fu’s F_S (Fu, 1997) was only recorded for dm 2,

indicating that only this deme is influenced by demographic

parameters (Table 1).

3.2. Genetic differentiation among demes

AMOVA analyses based on the five demes retrieved

by the BAPs analysis (Table 1) indicated that 87.47% of

the variation was due to differences among the demes, and 12.53% within the demes, with a highly significant

(p = 0.00000) F_ST value of 0.87 (Table 2). The pairwise

F_ST values were higher, ranging from 0.7, for dm 2 vs. dm 3, to 0.97 for dm 4 vs. dm 1 (Table 3).

The results of the Mantel test for the five demes

indicated a moderate degree of correlation between genetic and geographic distances (r = 0.54; p = 0.00001; Figure 4).

When dm 1 (r = -0.31; p = 0.19), dm 2 (r= -0.13; p = 0.79), and dm 5 (r= -0.04; p = 0.61) were analyzed separately the correlation were low, although dm 3 (r = 0.90; p = 0.001) and dm 4 (r = 0.89; p = 0.001) returned high correlations.

4. Discussion

Due to the ecological importance of D. albiventris, the investigation of the dynamics of dispersion of its populations through a genetic approach will improve the knowledge

and contribute to the establishment of management and conservation measures along its geographical distribution.

Were analyzed the largest sample for this specie to date, and high levels of haplotype diversity (h) and nucleotide

diversity (π) were found. Similar results were retrieved

in Sousa et al. (2012); h = 0.7235; π = 0.0065) for COI and Rocha et al. (2015) for Cytochrome b (h = 0.876;

π = 0.0062), reflecting the high genetic diversity of this

species. According to Grant and Bower (1998) high values of h and π combined (above 0.5 and 0.005 respectively)

Table 1. Genetic diversity and the results of the neutrality tests for the COI gene in the D. albiventris demes analyzed.

Demes Localities N NH Molecular diversity Neutrality tests

h π Test D Test Fs

Dm1 27 - 30 6 2 0.533 0.00087 0.85057 0.62543

Dm2 1 - 13, 16 e 17 40 6 0.529 0.00099 -1.14587 -2.65922*

Dm3 14, 15, 18, 19 e 25 18 5 0.614 0.00152 -1.13016 -1.43002

Dm4 19, 20, 23 e 25 20 2 0.479 0.00078 1.26176 1.31130

Dm5 20-26 58 4 0.387 0.00094 -0.72085 -0.38003

Pooled demes 142 19 0.846 0.00615 -0.17691 -0.50651

N: Sample size; NH: Number of haplotypes; h: Haplotype diversity; π: Nucleotide diversity; (*) = p < 0.05.

Table 3. Pairwise F_ST values for the COI gene for five demes of D. albiventris.

Deme Dm 1 Dm 2 Dm 3 Dm 4 Dm 5

Dm 1 0.00000

Dm 2 0.96492 0.00000

Dm 3 0.94811 0.71389 0.00000

Dm 4 0.97321 0.87873 0.85555 0.00000

Dm 5 0.96605 0.87873 0.81081 0.72012 0.00000

P = 0.0000.

Table 2. Results of the AMOVA for the COI gene for the five Didelphis albiventris demes retrieved from BAPs.

Source of variation Variance component % Variation F_ST p*

Among demes 2.18911 87.47 0.87465 0.0000

Within demes 0.31372 12.53

*Values of P, calculated randomly through 1023 permutations.

Figure 4. Results of the Mantel test for the correlation

between genetic (y) and geographic (x) distances in the COI

reflects long evolutionary history. In the present study our

results shows the haplotypes H11, H14 and H16 widely geographic distributed, suggesting long dispersion time and consequently long evolutionary history, corroborating

Grant and Bower (1998).

By randomly treating allelic frequencies of genetic marker and genetic divergence between groups, the BAPS

software retrieved five demes, which are divided between

the three regions analyzed, with overlap for demes 3 and 4 in the Northeast and Southeast Brazil. The BAPS was not unanimous in considering the geographic proximity or the biomes to delimit the demes, consequently other population analyzes were carried out to verify if they correspond to

different lineages.

In order to identify if there is selective neutrality, tests

were performed for the five demes. The results of Tajima’s D were not significant and Fu’s Fs was only significant

for dm 2, which contains haplotypes from the Northeast.

Significant results for these tests indicate the presence of

natural selection or demographic processes among the variants of a nucleotide sequence in a population. Dm 2 is

distributed over a wide area, with different kind of biomes

which could be interfering in the selective neutrality (Fu and Li, 1993; Nielsen 2001). No significant results were obtained for these same tests by Sousa et al. (2012) and Rocha et al. (2015), which indicates that this species is relatively constant. It is important to mention that these authors did not study northeastern populations.

The results of the AMOVA for the five demes indicate

structuring (F_ST = 0.87). This structuring is evident in the

case of dm 1, which has pairwise F_ST values of 0.95 to 0.97,

indicating possible processes of allele fixation and genetic

isolation. Sousa et al. (2012) also found evidence of population structuring (F_ST> 91%) in the Southeast and

South populations.

The remaining demes analyzed in the AMOVA

(dms 2, 3, 4 and 5) had also high pairwise F_ST values

(0.71 to 0.88). Despite the high F_ST, the same demes are

distributed in different localities, for example: dm 3 are in Caxias/MA and Xacriabá Amerindian Reservations/MG; dm 4 in Caxias/MA, Xacriabá Amerindian Reservations/MG, Belo Horizonte/MG and Nova Lima/MG; dm 5 in Xacriabá

Amerindian Reservations/MG, Belo Horizonte/MG

and Nova Lima/MG. We can also notice that different

demes are in sympatry, for example: dm 3 and dm 4 in

Caxias/MA; dm 3, dm 4 and dm 5 in Xacriabá Amerindian

Reservations/MG; dm 4 and dm 5 in Belo Horizonte/MG and Nova Lima/MG.

These sympatric demes show that F_ST are considering

different structured population in the same localities.

This condition could be explained by the wide distribution of D. albiventris (Figure 1) and by the highly adaptable and generalist habit of the specie, since it is known that altitude and environment do not interferes in the specie distribution (Aragona and Marinho-Filho, 2009). Thus, although F_ST shows structuring, the connection between demes allows that Northeastern and Southeastern regions belong to the same widespread lineages.

Studies of didelphids have shown population

structuring among different Brazilian biomes, for example Carvalho et al. (2011) and Caramaschi et al. (2011), found population structuring of Monodelphis domestica between the Cerrado and Caatinga. In the present study this

relationship was not verified, as the demes are not restricted to one specific biome and are dispersed among a number of different biomes. This fact may be associated in differences

in home ranges and in ecological characteristics between M. domestica and D. albiventris that could be possible

directly related with body size. Didelphis albiventris is the largest Didelphidae weighting 500-2,750g while M. domestica weight 80-120g only. This size difference

reflects in greater dispersion capacity (Reis et al., 2006; Wang et al., 2009) of D. albiventris that increases the gene

flow, making population structuring more difficult. In this

study, only dm 1 was isolated from the others, but this fact is not associated with biomes, since that deme occurs in Atlantic Forest and Pampa’s biomes.

When comparing the mutational sites among the demes, the haplotype network also shows two lineages

with different geographic distributions for D. albiventris

in this study: one Southern lineages (dm 1) and the other comprising Northeast/Southeast (demes 2-5) showed shared

haplotypes. These two principal lineages are separated by 15 mutational steps. Northeast and Southeast dm’s are

separated by few mutational steps (maximum of five, see

Figure 3), have distribution overlapping between biomes and therefore should be considered as a single lineage.

The gene flow shown in the haplotype network represents

an ancestral polymorphism, since the shared haplotypes are distributed over long geographic distances, as can be

seen in dm 3 (H11) that occurs in Rio Grande do Piauí/PI

(Cerrado) and Xacriabá Amerindian reservations/MG (Cerrado/Caatinga ecotone) (latitudes 07º 45’ S - 14° 53’ S). But the results of the haplotype network did not reveal

connection between dm 5 (H16 and18) in Piracema/MG (Atlantic Forest) and dm 1 (H1) Machadinho/RS (Atlantic Forest) which has similar latitude interval (latitudes 20º 30’ S - 27º 34’ S) (Figure 1 and 3; Table S1), another indication of the isolation of these two strains.

The Mantel test indicated a moderate degree of correlation between genetic and geographic distances in the

five demes. In this study, with the addition of D. albiventris samples from the Northeast, we obtained a reduction in the correlation obtained by Sousa et al. (2012). This reduction in the correlation reinforces the similarity found between demes 2-5, not diagnosed by these authors, and indicates that the Northeast/Southeast lineage is genetically similar although geographically distant.

Northeast and Southeast are connected. It is important to mention that connection of demes occurs in the transitional areas between biomes, which strengthens the conclusions of Sousa et al. (2012) and Rocha et al. (2015) about the importance of the transition zones for research and planning.

These areas are considered as hotspots of intraspecific

diversity and play an important role in the genetic diversity of species and should be closely investigated (Smith, 1997; Kark et al., 2002).

Our results provide important new insights into the population dynamics of small mammals in Brazil. It has long been assumed that these kind of mammals have low vagility, with geographically structured populations and highly divergent genetic traits (Crisci et al., 2003). The extensive connection between demes of the Northeast and Southeast for D. albiventris may reflect the ecological plasticity of this species, resembling a medium-large mammal, with large home-range.

In the present work we diagnosed two lineages

(Northeast/South axis) for the species D. albiventris and that the biomes do not interfere in the isolation of their

demes. However, as an essential finding, the present study

evidences extensive contact between demes at the transition zones, serving as reference to the conduction of important insights for the development of management actions.

Acknowledgements

We are grateful to Fundação de Amparo à Pesquisa e Desenvolvimento Científico do Maranhão (FAPEMA) for financing this study, and the students of the GENBIMOL

(Genetics and Molecular Biology Laboratory, Caxias,

Maranhão, Brazil) for their invaluable support in the field. This study was financed in part by the Coordenação de

Aperfeiçoamento de Pessoal de Nível Superior -Brasil

(CAPES) - Finance code 001.

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Supplementary Material

Supplementary material accompanies this paper.

Table S1. List of the D. albiventris specimens included in this study, their field numbers (BC, PR, FNI, ALN, TNM, T, PNSC, URC, LLLS, and DCN), GenBank accession number (JN), demes, haplotype number (H), locality,

geographical coordinates and origin of the samples.